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Candida peritonitis is a crucial disease, however the optimal antifungal therapy regimen has not been clearly defined. Peritoneal fibrosis (PF) can be caused by abdominal surgery, intra-abdominal infection, and malignant diseases, and is also widely recognized as a crucial complication of long-term peritoneal dialysis. However, the influence of PF on Candida peritonitis prognosis remains unknown. Here, we evaluated the severity of Candida peritonitis within the context of PF and the efficacy of micafungin using mice. A PF mouse model was generated by intraperitoneally administering chlorhexidine gluconate. Candida peritonitis, induced by intraperitoneal inoculation of Candida albicans, was treated with a 7-day consecutive subcutaneous administration of micafungin. Candida infection caused a higher mortality rate in the PF mice compared with the control mice on day 7. Proliferative Candida invasion into the peritoneum and intra-abdominal organs was confirmed pathologically only in the PF mice. However, all mice in both groups treated with micafungin survived until day 20. Micafungin treatment tends to suppress inflammatory cytokines in the plasma 12 h after infection in both groups. Our results suggest that PF enhances early mortality in Candida peritonitis. Prompt initiation and sufficient doses of micafungin had good efficacy for Candida peritonitis, irrespective of the underlying PF.

Candida albicans is a commensal fungus in human mucosal surfaces, including the oral cavity. Lactoferrin (LF) and the lactoperoxidase (LPO) system, which are host protection components in exocrine secretions, each exhibit weak anti-candida activity. We herein examined the effects of the combination of LF and the LPO system on C. albicans. Morphological observations indicated that the combination of LF and the LPO system reduced the mycelial volume of C. albicans and changed the size and shape of cells more than each agent alone. The combination of LF and the LPO system also exerted strong inhibitory effects on the cellular metabolic activity and adhesive hyphal form of C. albicans. A checkerboard analysis revealed that the anti-candida activity of LF and the LPO system was synergistic. These results suggest that the combination of LF and the LPO system is useful for preventing candidiasis.

Animal models of pathogenic infection are needed to evaluate candidate compounds for the development of anti-infectious drugs. Dermatophytes are pathogenic fungi that cause several infectious diseases. We established a silkworm dermatophyte infection model to evaluate anti-fungal drugs. Injection of conidia of the dermatophyte Arthroderma vanbreuseghemii into silkworms was lethal. A. vanbreuseghemii conidia germinated in liquid culture were more potent against silkworms than non-germinated conidia. Germinated conidia of other dermatophytes, Arthroderma benhamiae, Trichophyton rubrum, and Microsporum canis, also killed silkworms. Injection of heat-treated germinated A. vanbreuseghemii conidia did not kill silkworms, suggesting that only viable fungi are virulent. Injecting terbinafine or itraconazole, oral drugs used clinically to treat dermatophytosis, into the silkworm midgut had therapeutic effects against infection with germinated A. vanbreuseghemii conidia. When silkworms were injected with A. vanbreuseghemii expressing enhanced green fluorescent protein (eGFP), mycelial growth of the fungus was observed in the fat body and midgut. Injection of terbinafine into the silkworm midgut, which corresponds to oral administration in humans, inhibited the growth of A. vanbreuseghemii expressing eGFP in the fat body. These findings suggest that the silkworm infection model with eGFP-expressing dermatophytes is useful for evaluating the therapeutic activity of orally administered anti-fungal agents against dermatophytes.

Protamine peptide (PP) derived from salmon is a 14-mer with 10 arginine residues. We investigated the in vitro and in vivo antifungal activity of PP against Candida albicans PP showed a concentration-dependent dual mode of action, with fungicidal activity and inhibitory activity for hyphal development in vitro. At lethal concentrations of PP, intracellular accumulation of PP was energy-dependent but independent of endocytosis, and resulted in ATP efflux and the generation of reactive oxygen species in the cells. PP at sublethal concentrations inhibited hyphal development in C. albicans by binding to the cell surface. Though antifungal activity of PP was inactivated by high concentrations of NaCl, the antifungal activity of the synthetic cyclic (via a disulfide bond) form of PP (cyclic PP) was not. Cyclic PP also showed the concentration-dependent dual mode of action, and had five-fold greater antifungal activity than PP. The advantage of antifungal activity of cyclic PP compared with PP in vitro resulted in a high in vivo efficacy in a murine oral candidiasis model. Oral treatment with cyclic PP inhibited hyphal development of C. albicans on mouse tongues and protected against the development of severe candidiasis. This study shows the therapeutic potential of cyclic PP as an antifungal peptide against C. albicans., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A lysozyme-chitosan conjugate preparation (LYZOX), produced from egg white lysozyme and chitosan by Maillard reaction, is a commercial product developed as a cosmetic ingredient or food additive. Effects of LYZOX on in vitro growth of Candida albicans were examined. C. albicans cells were treated with LYZOX for 3 hrs, and then washed and cultured for an additional 16 hrs in modified RPMI1640 medium. Mycelial growth of C. albicans was clearly inhibited by more than 100 μg/ml of LYZOX in a concentration-dependent manner. On the other hand, corresponding concentration of chitosan or lysozyme or their mixture only scarcely showed clear inhibitory effect. Similarly, anti-Candida activity of the combination of LYZOX and decanoic acid, a middle-chain fatty acid, was also examined. Inhibitory activity of this combination against mycelial growth of C. albicans was very potent and appeared synergistic, since fractionated inhibitory concentration (FIC) index for 70% growth inhibition was calculated to be 0.20. Oral application of this combination improved the symptoms of Candida-infected-tongue in an experimental murine candidiasis model. On the basis of these results, the possible application of LYZOX as a new functional product with anti-candida activity was discussed.

The effects of ME1111, a novel antifungal agent, on the hyphal morphology and ultrastructure of Trichophyton mentagrophytes were investigated by using scanning and transmission electron microscopy. Structural changes, such as pit formation and/or depression of the cell surface, and degeneration of intracellular organelles and plasmolysis were observed after treatment with ME1111. Our results suggest that the inhibition of energy production by ME1111 affects the integrity and function of cellular membranes, leading to fungal cell death., (Copyright © 2016 American Society for Microbiology.)

Despite the existing treatment options for onychomycosis, there remains a strong demand for potent topical medications. ME1111 is a novel antifungal agent that is active against dermatophytes, has an excellent ability to penetrate human nails, and is being developed as a topical agent for onychomycosis. In the present study, we investigated its mechanism of action. Trichophyton mentagrophytes mutants with reduced susceptibility to ME1111 were selected in our laboratory, and genome sequences were determined for 3 resistant mutants. The inhibitory effect on a candidate target was evaluated by a spectrophotometric enzyme assay using mitochondrial fractions. Point mutations were introduced into candidate genes by a reverse genetics approach. Whole-genome analysis of the 3 selected mutants revealed point mutations in the structural regions of genes encoding subunits of succinate dehydrogenase (complex II). All of the laboratory-generated resistant mutants tested harbored a mutation in one of the subunits of succinate dehydrogenase (SdhB, SdhC, or SdhD). Most of the mutants showed cross-resistance to carboxin and boscalid, which are succinate dehydrogenase inhibitors. ME1111 strongly inhibited the succinate-2,6-dichlorophenolindophenol reductase reaction in Trichophyton rubrum and T. mentagrophytes (50% inhibitory concentrations [IC50s] of 0.029 and 0.025 μg/ml, respectively) but demonstrated only moderate inhibition of the same reaction in human cell lines. Furthermore, the target protein of ME1111 was confirmed by the introduction of point mutations causing the amino acid substitutions in SdhB, SdhC, and SdhD found in the laboratory-generated resistant mutants, which resulted in reduced susceptibility to ME1111. Thus, ME1111 is a novel inhibitor of the succinate dehydrogenase of Trichophyton species, and its mechanism of action indicates its selective profile., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Adherence is defined as the extent to which a person's behavior corresponds with recommendations from health care providers. Adherence to treatment is an important factor for a good therapeutic outcome., Objectives: This study aimed to examine the adherence of patients with tinea pedis and to clarify the factors related to it., Materials and Methods: We assessed medication adherence for oral and topical drugs using a translated version of the Morisky Medication Adherence Scale-8 (MMAS-8) together with other background factors in 445 Japanese patients with tinea pedis, using a questionnaire in a web-based monitoring system., Results: Overall, high, medium and low adherence rates as assessed by MMAS-8 were 8.7%, 31.7% and 59.6% for oral medication, and 8.6%, 17.4% and 74.0% for topical medication, respectively. The adherence level was significantly higher for oral medication than for topical medication. Subgroup analyses showed that the adherence level for topical medication was significantly higher when topical and oral medications were used in combination than when topical medication was used alone. A low adherence level was shown in employed patients, those for whom their oral medication had not been effective and those with topical medication who had visited their hospital less often than once every six months., Conclusion: Patient adherence to therapy can be effectively improved by selecting highly effective medication while considering the prescription of topical and oral antifungal medications concomitantly, by carefully selecting a therapy plan for employed patients and by encouraging patients to visit their doctor regularly.

Micafungin (MCFG) concentrations in the plasma and in burn eschar were investigated after daily intravenous infusion (1 h) of MCFG (200 mg) in three patients with severe burns. MCFG treatment was initiated more than 72 h after the burn injuries. The MCFG concentrations in the plasma were determined at the end of the first administration of MCFG, immediately before the second dosing, at the end of the MCFG infusion after at least 4 days from the initial treatment, and immediately before the subsequent dosing using high-performance liquid chromatography. In addition, the trough levels in burn eschar after both the initial administration and repeated administration were measured. The peak and trough levels in the plasma were comparable to or slightly lower than the reported values in healthy volunteers. The mean (range) MCFG concentrations in the burn eschar after initial administration and repeated administration were 1.41 μg/mL (<0.1-3.98 μg/mL) and 6.65 μg/mL (1.10-14.81 μg/mL), respectively. In most cases, the MCFG concentrations in the burn eschar, especially after repeated administration, were higher than the reported MIC90 of MCFG against the clinically important pathogenic species of Candida and Aspergillus. These results suggest that MCFG is capable of penetrating burn eschar.

The combined effect of terpinen-4-ol, the main component of tea tree oil, and capric acid against mycelial growth of Candida albicans and murine oral candidiasis was evaluated in vitro and in vivo. Mycelial growth of C. albicans was estimated by the Cristal violet method. Combination of these compounds revealed a potent synergistic inhibition of growth. Therapeutic efficacy of the combination was evaluated microbiologically in murine oral candidiasis, and its application of the compounds clearly demonstrated therapeutic activity. Based on these results, the combined agent of terpinen-4-ol and capric acid was discussed as a possible candidate for oral candidiasis therapy.

We assessed anti-C. albicans activities of the 4 fatty acids : caproic acid, caprylic acid, capric acid and lauric acid in vitro. All four inhibited not only the mycelial but also the yeast-form growth of Candida albicans. In particular, capric acid and caprylic acid inhibited Candida mycelia growth at very low concentrations. The effects of treatment of these two fatty acids on oral candidiasis were examined using a murine model. When 50 µl of capric acid (more than 48.8 µM) was administered three times into the oral cavity of Candida-infected mice, symptom scores of tongues of the mice were significantly improved. Histological studies of the capric acid-treated animals indicated that the fatty acid suppressed mycelial growth of the fungus on the tongue surface. These results suggest that all four fatty acids, and especially capric acid, have potential as substances supporting anti-Candida treatment.

The therapeutic efficacy of tea tree oil (TTO), Melaleuca alternifolia, and its main component, terpinen-4-ol, were evaluated in a murine oral candidiasis model. Prednisolone -pretreated mice were orally infected with a fluconazole-susceptible (TIMM 2640) or a resistant (TIMM 3163) strain of Candida albicans to induce oral candidiasis. TTO or terpinen-4-ol was administrated with a cotton swab 3 h and 24 h after candida infection. These treatments clearly showed a decrease in the symptom score of tongues and in the viable candida cell number in the oral cavity at 2 d after azole-susceptible C. albicans infection, although the degree of the efficacy was less than that of fluconazole. Even against oral candidiasis caused by azole-resistant C. albicans, TTO and terpinen-4-ol were similarly effective, while fluconazole appeared ineffective. These results suggest that TTO and terpinen-4-ol may have the potential of therapeutic ability for mucosal candidiasis which may also be applicable to C. albicans oral candidiasis induced by the azole-resistant strain.

To evaluate the effect of the thiocarbamate antifungal agent liranaftate on inflammation and itchiness, footpad edema by phorbol 12-myristate 13-acetate (PMA) and the paw-licking accompanying by perceptual stimuli by compound 48/80 were examined. The effect of liranaftate application to mouse footpad on paw-licking time by compound 48/80 was observed. Topical administration of 4% liranaftate 1 hr before compound 48/80 did not suppress the paw-licking time, while pyrilamine, an anti-histamine agent, suppressed it significantly. As liranaftate was reported to suppress the ear inflammation induced by PMA, the effect of this agent on the footpad edema by PMA was examined. Liranaftate application significantly suppressed the increase in footpad swelling 24 hr after application of PMA, as true with ear inflammation. In this condition, we measured the paw-licking time by compound 48/80, but the suppression of time was not observed by the agent with or without the suppression of footpad inflammation. From these observations, we conclude that liranaftate treatment suppresses late phase inflammatory reaction in feet, perhaps accompanied by cytokine production, though it may not relieve acute stimuli and itchiness through an anti-histamine effect directly.

E1210 is a new antifungal compound with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clinical fungal isolates. E1210 showed potent activities against most Candida spp. (MIC(90) of ≤0.008 to 0.06 μg/ml), except for Candida krusei (MICs of 2 to >32 μg/ml). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC(90) of 0.13 μg/ml). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 μg/ml), Scedosporium prolificans (MIC of 0.03 μg/ml), and Paecilomyces lilacinus (MICs of 0.06 μg/ml) among the compounds tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been observed with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.

E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action-inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210's potential for the treatment of disseminated fungal infections are indicated.

HPLC/MS analysis revealed that the main constituents of Brazilian propolis A and B were artepillin C and drupanin, while those of New Zealand propolis C were pinocembrin, galangin and alkylphenol. No flavonoid or phenolic acid was detected in Japanese propolis D. Propolis C showed comparatively potent activity against growth of Trichophyton mentagrophytes, against filament formation of Candida albicans and potent scavenging activity against 1,1-diphenyl-2-picrylhyrazyl radical, but was less active against growth of C. albicans, as compared with those of thyme thymol essential oil, which was used as a positive control. Propolis A, B, and D were weak in antifungal activity, but showed more potent radical scavenging activity than thyme thymol oil. These results reveal the unique bioactivity of propolis, suggesting a possible application for antifungal therapy.

Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro antifungal activity against fungi. These synthesized amides have potent antifungal activity against Candida albicans and Aspergillus fumigatus. Our results suggest that hetero ring amides may be potent antifungal agents that operate by inhibiting the function of Gwt1 protein in the GPI biosynthetic pathway., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The Japanese Society for Medical Mycology (JSMM) method used for testing the antifungal susceptibility of yeast, the MIC end point for azole antifungal agents, is currently set at IC(80). It was recently shown, however that there is an inconsistency in the MIC value between the JSMM method and the CLSI M27-A2 (CLSI) method, in which the end- point was to read as IC(50). To resolve this discrepancy and reassess the JSMM method, the MIC for three azoles, fluconazole, itraconazole and voriconazole were compared to 5 strains of each of the following Candida species: C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei, for a total of 25 comparisons, using the JSMM method, a modified JSMM method, and the CLSI method. The results showed that when the MIC end- point criterion of the JSMM method was changed from IC(80) to IC(50) (the modified JSMM method) , the MIC value was consistent and compatible with the CLSI method. Finally, it should be emphasized that the JSMM method, using a spectrophotometer for MIC measurement, was superior in both stability and reproducibility, as compared to the CLSI method in which growth was assessed by visual observation.

We assessed the potential of oligonol, a low molecular polyphenol formulation prepared from lychee fruits, for treatment of oral candidiasis using a murine model. Oligonol at concentration of more than 313 microg/ml inhibited the mycelial growth of Candida albicans in vitro. When 50 microl of oligonol (20 mg/ml ) was administered three times into the oral cavity of orally Candida -infected mice, the number of viable Candida cells in the oral cavity was reduced significantly and the score of lesions on the tongue recovered on day 2. These findings suggest that oligonol could have potential as a food component supporting anti- Candida treatment.

To evaluate the anti-inflammatory activity of the thiocarbamate antifungal agent liranaftate, the edema and the neutrophil accumulation detected by the activity of neutrophil marker enzyme, myeloperoxidase (MPO), were examined following application of liranaftate to mouse ears with inflammation induced by phorbol 12-myristate 13-acetate (PMA). Topical 20 microl administration of liranaftate in a dose-range between 1-4% suppressed the increase in ear thickness 6 hr after PMA application dose-dependently. Similarly, it decreased the weight increase of an ear section after 24 hr dose-dependently. More than 1% of liranaftate also suppressed augmentation of MPO activity of the ear section. This and histological observation indicate that liranaftate treatment suppressed neutrophil accumulation in PMA-applied ear lesion. From these results, we discussed that liranaftate might suppress inflammatory symptoms caused by trychophytosis in a clinical condition.

Farnesol is known as a quorum-sensing molecule for Candida albicans and is recognized to play pathogenic roles in Candida infection. To assess the possible role of farnesol in mucosal C. albicans infection, the effects of farnesol treatment against experimental oral candidiasis in mice were examined. Prednisolone-pretreated ICR mice were orally infected with C. albicans and 3, 24 and 30 hr later the animals were orally given farnesol. Forty-eight hr later they were killed for observation. Farnesol treatment in a dose ranging between 1.125 and 9 micromol/mouse showed a protective effect against oral candidiasis in a dose-dependent manner, at least as estimated by symptom scores of tongues. At 9 micromol/mouse it decreased bodyweight loss. Histological studies of 2.25 micromol/mouse farnesol-treated animals indicated that farnesol suppressed mycelial growth of C. albicans on the surface of tongues, but microbiological study did not prevent the change of CFU of C. albicans cells not only on tongues but also in feces, kidneys and livers. These results suggest that farnesol has very characteristic roles in protection against mucosal candidiasis.

After the rapid progress in therapeutic pharmaceutics against onychomycosis caused by dermatophytes in the 1990s, an optimal therapeutic strategy for individual patients with the onychomycosis has become possible for clinical dermatologists. In this review, we discuss on clinical problems concerning this disease and propose recommendable treatments for each patient with topical and/or systemic use of antifungal agents. Finally, with consideration of already published therapeutic guidelines, we stress the necessity of "order-made" therapy for each patient with his/her medical status and wishes taking into account.

Objective: The therapeutic activities of cyclodextrin-associated itraconazole oral solution (itraconazole OS) by two administration routes in experimental oral and oesophageal candidiasis in mice were examined and compared., Methods: Using experimental oral and oesophageal candidiasis models in ICR mice, we investigated the efficacy of oral and intragastric administration of itraconazole OS and checked the concentration of itraconazole and its metabolite hydroxyitraconazole (OH-itraconazole) in tongues or oesophagus tissue after administration of itraconazole OS., Results: Oral administration of itraconazole OS at doses of 0.8, 4.0 or 20 mg/kg/day clearly decreased the number of viable Candida albicans cells in the oral cavity of mice with oral candidiasis in a dose-dependent manner at 3 days after infection. Intragastric administration of itraconazole OS at doses of 4 and 20 mg/kg/day once a day were also effective but to a lesser degree than that of oral administration. In the oesophageal candidiasis model, oral administration of itraconazole OS displayed superior therapeutic efficacy to the intragastric route. In coincidence with the greater efficacy, itraconazole was detected in lesional tissues after oral administration of itraconazole OS., Conclusions: Oral administration of itraconazole OS displayed therapeutic efficacy against murine oral and oesophageal candidiasis superior to that achieved by intragastric administration. This can be explained by there being higher concentrations of itraconazole in tongues or oesophagus tissues after administration of the suspension by the oral route.

The serotypes and mating types of 80 clinical isolates of Cryptococcus neoformans from Kenya were studied and subjected to broth microdilution susceptibility testing to amphotericin B (AMP), flucytosin, fluconazole (FLC), itraconazole (ITC) and miconazole (MCZ). The isolates included C. neoformans var. grubii- 75 of 80 (serotype A; 93.7%), C. neoformans var. neoformans- three of 80 (3.8%) and C. neoformans var. gattii- two (serotype B; 2.5%). Mating experiment confirmed all the isolates to be alpha-mating type. Seventy-eight (97.5%) of the isolates had minimum inhibitory concentration (MIC) of < or =0.5 microg ml(-1) to AMP and at 1 microg ml(-1), 100% of the isolates were inhibited. Flucytosin resistance was observed in 21% with MIC in which 90% of the isolates were inhibited (MIC90) of 64 microg ml(-1). Only 23.8% of the strains were susceptible to FLC with 65% susceptible dose-dependent (SDD) and 11.2% resistant. Itraconazole susceptibility was 61.3% while the rest were either SDD or resistant. The MIC90 for ITC and MCZ were 0.5 and 2 microg ml(-1) respectively. The study reports the serotypes, mating types and highlights the existence of azoles resistance in C. neoformans in Nairobi which calls for antifungal drug resistance surveillance as prophylactic use of FLC increases because of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic in sub-Saharan Africa.

The efficacy of itraconazole (ITZ) solubilized in hydroxypropyl-beta-cyclodextrin (ITZ-IV) was examined in a murine model of invasive pulmonary aspergillosis (IPA). Immunosuppressed mice were infected by the intratracheal inoculation of Aspergillus fumigatus conidia (2 x 10(6) conidia/mouse). Their body weight rapidly decreased and they died within 6 days after infection. Intravenous administration of various doses of ITZ-IV was started 24 h after infection and was continued once a day for 4 days. ITZ-IV at daily doses of 10, 20, or 40 mg/kg was as effective as the intraperitoneal administration of amphotericin B (AMPH) at a dosage of 1 mg/kg daily in improving survival. ITZ-IV (20 mg/kg per day), as well as AMPH (1 mg/kg per day) significantly lowered the fungal burden in the pulmonary tissues. Histological improvement was seen within 2 days after the beginning of administration of ITZ-IV (20 mg/kg per day). In mice intravenously given a single dose of ITZ-IV (20 mg/kg), the blood level and pulmonary tissue level of ITZ plus its active metabolites, mainly hydroxyitraconazole (OH-ITZ), decreased gradually after the injection, but after 4 h their concentration was still between 1.4 microg/ml (ITZ) and 1.9 microg/ml (OH-ITZ), concentrations that were approximately 10 to 20 times greater than the minimum inhibitory concentration (MIC) of ITZ for challenging the strain of A. fumigatus (0.16 microg/ml). These results support the clinical usefulness of ITZ-IV for the treatment of IPA in immunocompromised patients.

The vapor activity of six essential oils against a Trichophyton mentagrophytes was examined using a closed box. The antifungal activity was determined from colony size, which was correlated with the inoculum size. As judged from the minimum inhibitory dose and the minimum fungicidal dose determined after vapor exposure for 24 h, the vapor activity of the six essential oils was ranked in the following order: oregano > clove, perilla > geranium, lavender, tea tree. The vapors of oregano, perilla, tea tree, and lavender oils killed the mycelia by short exposure, for 3 h, but the vapors of clove and geranium oils were only active after overnight exposure. The vapor of oregano and other oils induced lysis of the mycelia. Morphological examination by scanning electron microscope (SEM) revealed that the cell membrane and cell wall were damaged in a dose- and time-dependent manner by the action of oregano vapor, causing rupture and peeling of the cell wall, with small bulges coming from the cell membrane. The vapor activity increased after 24 h, but mycelial accumulation of the active oil constituents was maximized around 15 h, and then decreased in parallel with the decrease of vapor concentration. This suggested that the active constituent accumulated on the fungal cells around 15 h caused irreversible damage, which eventually led to cellular death.

For practical application for vapor therapy and fumigation, the vapor activity of 72 essential oils was screened against a Trichophyton mentagrophytes, using a closed box. The fungicidal activity, expressed as the minimum fungicidal dose (MFD), was determined from the colony size, which was correlated with the inoculum size. Oils containing phenol as the major component showed the most potent vapor activity, with an MFD of 1.56 microg/ml air. This was followed by oils with aldehyde as the major component, then by those with alcohol as the major component. The vapor activities of oils containing ketone, ester, and ether/oxide components were decreased, in that order. The oils that were rich in hydrocarbon components had the weakest activity. The same tendency was observed with the components themselves. Phenols and aldehydes exhibited the highest vapor activity, followed by alcohols, ketone, ester, ether/oxide, and hydrocarbon. There was a rough correlation between the vapor activity determined by the box vapor assay and the contact activity determined by agar diffusion assay. But oils containing sesquiterpenes showed weaker vapor activity than that expected from the contact activity. Based on the activity-chemical structure relationship, the 72 oils were classified into seven functional groups.

The antimicrobial activity against two bacterial strains and three fungi of five terpenoid quinones, three related quinones, and a quinone-containing essential oil was investigated by broth dilution and by agar vapor and box vapor assays. All the quinones, except for coenzyme Q10, exhibited their highest activity against Trichophyton mentagrophytes. Thymoquinone showed the most potent activity, not only by solution contact but also by vapor contact, with menadione the next most potent. Alizarin showed high activity only by solution contact. The antibacterial activity was related to the orthoquinone structure (1,2-chloranil and 1,2-naphthoquinone) and the antifungal activity was related to the paraquinone structure (chloranil, ubiquinone 0, thymoquinone, and menadione).

To study the postantifungal effect (PAFE) of itraconazole (ITCZ) against Candida species, we quantitated it using a newly introduced parameter T/C(T=time required for the drug-treated culture to reach 5-fold increase in turbidity; C=time required for the drug-free control culture to reach 5-fold increase in turbidity) referred as PAFE index and compared the results with those obtained for two other azole antifungal drugs fluconazole (FLCZ) and miconazole (MCZ). The mean values of PAFE index for ITCZ against three C. albicans strains were 1.28, 1.45, and 1.60 when exposed to the drug for 1 hr at concentrations of 1 x MIC, 2 x MIC and 4 x MIC, respectively. These values are similar to those for MCZ and appreciably higher than those for FLCZ. PAFE index values for ITCZ against each single strain of three non-albicans Candida species exposed to 2 x MIC of the drug for 1 hr were: 1.22 with C. glabrata, 1.63 with C. parapsilosis, and 3.90 with C. tropicalis. Again, comparable values were obtained for MCZ and lower values for FLCZ. The drug concentration for exposure and the duration of exposure only slightly or scarcely affected the PAFE of ITCZ, although the magnitude of the PAFE considerably varied among different strains or species tested. In general, ITCZ, as well as MCZ, produced greater extents of PAFE than those for FLCZ against C. albicans and several non-albicans Candida species.

By the combined use of agar diffusion, agar vapor and agar vapor-inhibitory assays, contribution of the vapor activity of essential oils was quantitatively estimated. The test organisms were Trichophyton mentagrophytes and Aspergillus fumigatus. Agar vapor assay was used to confirm the vapor activity of the oils. The parameter delta defined as a contribution index of the vapor activity was calculated by (1 - b-c/a-c) x 100, where a is inhibitory diameter in the diffusion assay, b is inhibitory diameter in the vapor-inhibitory assay and c is diameter of the sealed ring in the vapor-inhibitory assay (21 mm). Many of the essential oils examined showed a delta value near 100, thus providing the major contribution of the vapor activity to the inhibitory diameter. Essential oils containing aldehyde as major constituent showed low delta value, indicating the major inhibition was due to agar diffusion. Major essential oil components behaved similarly; the delta value was increased in the following order: aldehyde < phenol < alcohol < ester, oxide, hydrocarbon, indicating the enhanced contribution of the vapor activity in that order.

We examined the effect of a clove (Syzygium aromaticum) administered by two different routes on Candida albicans growth, using a murine oral candidiasis model. When the clove preparation was administered into the oral cavity of Candida-infected mice, their oral symptoms were improved and the number of viable Candida cells in the cavity was reduced. In contrast, when the clove preparation was administered intragastrically, oral symptoms were not improved, but viable cell numbers of Candida in the stomach and feces were decreased. These findings demonstrate that oral intake of an herbal food, clove, may suppress the overgrowth of C. albicans in the alimentary tract including the oral cavity.

The effects of picolinic acid (PLA), a product of tryptophan catabolism, on anti- Candida activity of neutrophils were studied. Casein-induced peritoneal neutrophils of C3H/He mice partially inhibited mycelial growth of Candida albicans when cultured with C. albicans for 16 h in vitro. The growth inhibition of Candida was augmented by a combination of neutrophils and more than 4 mM picolinic acid. Especially in the presence of 200 U/ml murine interferon-gamma (IFN-gamma), 2 mM picolinic acid augmented the anti- Candida activity of neutrophils. The physiological significance of the augmenting effects of picolinic acid is discussed.

Therapeutic activity against oral candidiasis of orally administered bovine lactoferrin (LF), a multifunctional milk protein, was shown in a previous report using an immunosuppressed murine model. In the present study, the influence of orally administered LF on immune responses relevant to this therapeutic effect was examined. Because mice were immunosuppressed with prednisolone 1 day before and 3 days after the infection with Candida, the numbers of peripheral blood leukocytes (PBL) and cervical lymph node (CLN) cells were reduced. LF feeding prevented the reduction in the numbers of PBL on day 1 and CLN cells on days 1, 5 and 6 in the Candida-infected mice. The number of CLN cells of individual mice on days 5 and 6 was inversely correlated with the Candida c.f.u. in the oral cavity. Increased production of IFN-gamma and TNF-alpha by CLN cells stimulated with heat-killed Candida albicans on day 6 was observed in LF-treated mice compared with non-treated mice. Concanavalin A (ConA)-stimulated CLN cells from LF-treated mice also showed a significant increase in the production of IFN-gamma and IL12 on day 5 and a tendency for increased production of IFN-gamma and TNF-alpha on day 6. The levels of cytokine production by ConA-stimulated CLN cells on day 6 were inversely correlated with the Candida c.f.u. in the oral cavity. In conclusion, the alleviation of oral candidiasis by LF feeding in this model may correlate with the enhancement of the number of leukocytes and their cytokine responses in regional lymph nodes against Candida infection.

Some types of antimicrobial agents such as fosfomycin, clarithromycin, and erythromycin have been known to affect the host's immunological response. We investigated whether itraconazole (ITCZ) can modulate the serum cytokine levels (tumor necrosis factor (TNF-alpha, interleukin (IL)-10, IL-12p40) in both patients and healthy subjects. Serum samples were collected and TNF-alpha, IL-10 and IL-12p40 were measured using a sandwich enzyme-linked immunosorbent assay (ELISA). We showed increased levels of IL-12p40 in patients treated with ITCZ. In healthy subjects, moreover, ITCZ treatment significantly increased the production of IL-12p40, but not TNF-alpha or IL-10. These findings suggest that the effectiveness of ITCZ in the treatment of fungal infection may be due not only to its direct antifungal effect, but also to induction of an immunomodulative effect on the host defense systems by regulation of cytokine production.

Prophylactic effects upon imunnosuppressed mice lethally infected by Candida albicans were examined in fractions prepared from a constituent herb of Juzen-taiho-to (TJ-48, Si-Quan-Da-Bu-Tang), rhizomes of Atractylodes lancea DC. The oral administration of water extract obtained from a residue after MeOH extraction of rhizomes significantly prolonged the survival period of the infected mice at a dose of 140 mg/kg/day compared with control mice, while the MeOH extract did not. In the crude polysaccharide fraction (F-2) obtained by EtOH precipitation of the water extract, a significant life-prolonging effect was observed by the administration of 70 mg/kg/day. F-2 was further fractionated, and the resulting strongly acidic polysaccharide fraction, F-2-2, had a protective effect at a dose of 17.5 mg/kg/day. This fraction mainly consisted of acidic pectic polysaccharides containing about 80% galacturonic acid. The protective activity of F-2-2 was lost by periodate oxidation, but not by protease digestion, suggesting that the polysaccharide component of F-2-2 plays a major role in the protective activity against Candida-infected mice.

Synergism of lysozyme with the imidazole antifungal lanoconazole (LCZ) has been demonstrated in vitro against Candida albicans. To determine the mechanism of the synergistic action, the effects of subinhibitory concentrations of lysozyme (50 microg ml(-1)) and LCZ (1.25 microg ml(-1)), used alone and in combination, on the morphology and ultrastructure of C. albicans cells were studied by fluorescence microscopy using Fungiflora Y, as well as by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In the presence of lysozyme, the separation of apparently mature cells from each other was inhibited, and cells in which cell wall-like material had markedly accumulated in the periplasmic space were frequently observed. Such intracellular ultrastructural changes were not seen in LCZ-treated cells, but normal septal wall biogenesis was inhibited with the resultant formation of long chains of interconnecting cells. When the cultures were treated with the combination of lysozyme and LCZ, intracellular accumulation of cell wall-like materials increased in extent, and the peripheral wall structure was severely damaged. These morphological and ultrastructural findings lead us to the following conclusions: (1) lysozyme and LCZ inhibit the process of cell division in different manners; (2) lysozyme appears to affect cell wall formation more directly and drastically by interrupting the normal assembly and integration of cell wall components; and (3) these effects of lysozyme are noticeably enhanced when combined with LCZ.

Itraconazole (ITCZ), a triazole antifungal agent, was studied for its effects on the morphology and function of L929 fibroblasts. L929 fibroblasts were cultured for 20 h with ITCZ or one of several other triazoles (fluconazole, ketoconazole, and hydroxy-itraconazole [ITCZ-OH]) at the concentration of 0.5 microg/ml. Among these agents, only ITCZ and its metabolite ITCZ-OH markedly elongated the cells bidirectionally. Scanning electron microscopy studies showed that the surface of the elongated cells was smoother than that of the untreated cells. The viability of L929 cells cultured with 0.5 microg/ml of ITCZ for 20 h was not lowered. However, after treatment with 0.0375% sodium deoxycholate (DOC) solution, the viability of the cells treated with ITCZ, as evaluated by the 3-(4,5-dimethyl-2thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay or the release of lactic dehydrogenase from cytoplasm, was decreased. When L929 cells were cultured in the presence of a combination of ITCZ and vincristine, their growth was synergistically inhibited. This synergism was also observed when ITCZ was replaced by ITCZ-OH, but not by the other azoles. These findings suggest that the exposure of L929 fibroblasts to low ITCZ concentrations affects the physiological nature of their cell membrane.

The anti-inflammatory effect of lanoconazole (LCZ) was investigated in vivo and in vitro. The effect of LCZ was evaluated on the inflammatory reactions elicited by intradermal injection of ovalbumin to ovalbumin-immunized rabbits, as an Arthus phenomenon. A one or two % cream preparation of LCZ was topically applied on the lesion daily after challenging injection until the inflamation had diminished. By macroscopic observation and measuring the diameter of edema, erythema, hemorrhage and necrosis, the effects of LCZ on the reactions were compared with the reactions of the sites administered withcream vehicle as reference agent. Two % LCZ showed an anti-hemorrhagic effect. The in vitro effect of LCZ on production and functions of an inflammatory cytokine, TNF was also examined. LCZ suppressed the production of TNF by murine peritoneal macrophages at 20 micro g/ml and the adhesion of neutrophils at 100 micro g/ml. Moreover, LCZ significantly suppressed the growth inhibitory activity of TNF against L929 fibroblasts at 0.5 micro g/ml. A very low concentration of LCZ might protect the fibroblasts from immunological cytotoxicity in vivo. These findings suggest that LCZ has a suppressive activity to inflammatory responses and this suppressive action may be due to its protective activity to cells like fibroblasts.

The effects of bovine lactoferrin (LF) or the LF-derived antimicrobial peptide lactoferricin B (LFcin B) on the growth of Candida albicans hyphae, including those of three azole-resistant strains, were investigated by a crystal violet staining method. The hyphae of two highly azole-resistant strains were more susceptible to inhibition by LF or LFcin B than the azole-susceptible strains tested. One moderately azole-resistant strain was defective in the formation of hyphae and showed a susceptibility to LF greater than that of the susceptible strains but a susceptibility to LFcin B similar to that of the susceptible strains. The highly azole-resistant strain TIMM3317 showed trailing growth in the presence of fluconazole or itraconazole, while the extent of growth was reduced by the addition of LF or LFcin B at a sub-MIC. Thus, the addition of LF or LFcin B at a sub-MIC resulted in a substantial decrease in the MICs of fluconazole and itraconazole for two highly azole-resistant strains; e.g., the MIC of fluconazole for TIMM3317 was shifted from > 256 to 0.25 micrograms/ml by LF, but the MICs were not decreased for the susceptible strains. The combination effects observed with triazoles and LF-related compounds in the case of the two highly azole-resistant strains were confirmed to be synergistic by the fractional inhibitory concentration index. These results demonstrate that for some azole-resistant C. albicans strains, LF-related compounds combined with triazoles can inhibit the growth of hyphae, an important form of this organism in pathogenesis.

Morphological effects of itraconazole (ITCZ) on murine macrophages were examined by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and compared with the effects of other antifungal agents. Thioglycolate-induced peritoneal macrophages were prepared from C3H/He J mice and cultured for 20 h in the presence of the antifungal azoles econazole, fluconazole, miconazole, ketoconazole, ITCZ, hydroxy-itraconazole (ITCZ-OH), and a polyene antibiotic amphotericin B (AMPH). Among these reagents, only ITCZ and its derivative ITCZ-OH were effective in causing morphological changes of murine macrophage as determined by LM and SEM. Macrophages treated with 2 microg/ml ITCZ or ITCZ-OH were stretched out bidirectionally, their surface was smooth and their 'ruffles' decreased. TEM observation showed that the bundles of the filamentous structure existed along the cell shape in the cytoplasm. These findings suggest that ITCZ and ITCZ-OH affect the morphology of macrophages.

The effect of an anti-microbial protein, calprotectin, in combination with neutrophils on the growth of Candida albicans was investigated. The growth inhibition of C. albicans by murine neutrophils was augmented by the addition of a low concentration of calprotectin prepared from rat peritoneal exudate cells. The concentrations of calprotectin causing 50% inhibition of growth of C. albicans in the absence or presence of neutrophils at an effector-to-target (E/T) ratio of 30 and 60 were estimated to be 0.45, 0.34 and 0.28 U/ml, respectively. The anti-Candida activity of calprotectin was completely inhibited by 2 microM of zinc ion, while it only partially lowered the activity of the combination of calprotectin and neutrophils. Lactoferrin, which is an anti-microbial protein released from neutrophils, strongly inhibited the growth of C. albicans in combination with calprotectin. These results suggest that calprotectin and lactoferrin released from neutrophils may cooperate to inhibit the growth of C. albicans at a local lesion of the infection where there is an accumulation of neutrophils.

We investigated the effects of lactoferrin (Lf)-related compounds on growth inhibition of Candida albicans by neutrophils or antifungal agents in vitro. Human neutrophils partially inhibited the growth of C.albicans. The growth inhibition caused by human neutrophils was augmented by the addition of human Lf at concentrations which did not show any inhibitory effect in the absence of neutrophils. Similar observations were obtained also with the following combinations: human neutrophils + bovine Lf, murine neutrophils + bovine Lf, and murine neutrophils + iron saturated bovine Lf, but not in the case of murine neutrophils + human transferrin. The minimum inhibitory concentration (MIC) of azole antifungal agents, clotrimazole, ketoconazole, fluconazole, and itraconazole was reduced by 1/4 to 1/16 in the presence of a sub-MIC level of each of bovine Lf, bovine Lf pepsin hydrolysate, and the antimicrobial peptide "lactoferricin B" (Lfcin B). Other types of antifungal agents, amphotericin B, nystatin, and flucytosine did not show such combined effects with these Lf-related compounds. The anti-Candida activity of bovine Lf or Lfcin B in combination with clotrimazole was shown to be synergistic by checkerboard analysis. Clinically isolated azole-resistant C. albicans strains were more susceptible to bovine Lf or Lfcin B than azole-susceptible strains. Trailing growth of an azole-resistant strain in the presence of fluconazole was reduced by the addition of sub-MIC levels of bovine Lf or Lfcin B. These results suggest that Lf-related compounds even at relatively low concentrations may function as an antifungal effector in combination with neutrophils thereby modulating azole antifungal efficacies in vivo.

Effects of itraconazole (ITCZ) on anti-Candida activity of a macrophage (MP)-neutrophil axis were examined in vitro. Murine peritoneal MP, stimulated by an enterococcal preparation FK-23, released tumor necrosis factor a (TNF) into the culture medium in 20 hr-culture period. This level of TNF production by the stimulated MP, which were measured by a bioassay or immunological assay, was heightened by co-stimulation with 0.5 mg/ml of ITCZ. Supernatants obtained from the culture medium of FK-23 and ITCZ-treated MP not only contained about 10 ng/ml of TNF but also had the capacity to enhance anti-Candida activity of murine neutrophils. This enhancement was blocked by pretreatment of the MP-supernatant with anti-TNF antibody. Observation by scanning electron microscope suggested that cell walls of the candida mycelia, the growth of which was inhibited by the neutrophils in the presence of either the MP-supernatant or TNF, sustained similar structural damage. These results suggest that ITCZ augments anti-Candida activity of neutrophils through enhanced production of TNF by MP in vitro.

The combined effects of lysozyme and various antifungal agents were investigated by microbroth dilution method against Candida albicans in vitro. Synergistic anti-Candida activity was observed between egg white lysozyme and itraconazole, clotrimazole, miconazole and lanoconazole. Similar, although not as clear, combination anti-Candida activities were seen in the cases of ketoconazole, bifonazole, amphotericin B and nystatin. Anti-Candida activity of fluconazole was not affected by the addition of lysozyme, however. Physiological roles of this combination effect in anti-Candida therapy by azole antifungals were discussed.

Benanomicin A is an antifungal antibiotic produced by Actinomadura spadix. In the present study, we investigated the effect of benanomicin A on the phagocytosis of Candida albicans by murine peritoneal macrophages and on the cell-surface hydrophobicity (CSH) of C. albicans. Although pretreatment of macrophages with benanomicin A had no effect on the phagocytosis, addition of benanomicin A to the culture of macrophages and Candida cells increased the susceptibility of Candida cells to the phagocytosis by the macrophages. Pretreatment of Candida cells with benanomicin A also increased the susceptibility of Candida cells to the phagocytosis. When Candida cells were mixed with benanomicin A, the antibiotic bound irreversibly to Candida cells. These data suggest the possibility that the increased susceptibility of Candida cells to the phagocytosis is mediated by the binding of benanomicin A to Candida cells. Examination of physicochemical property of Candida cell surface showed that the CSH of Candida cells significantly decreased by the treatment with benanomicin A. Thus, binding of benanomicin A to Candida cells may induce biochemical/physicochemical alternation of the surfaces, so that they become more susceptible to phagocytosis by murine macrophages. These properties of benanomicin A, along with its antifungal activity, seem to be beneficial in the treatment of fungal infections.

The effects of lactoferrin (LF), an antimicrobial protein secreted in body fluids, and its peptides in combination with azole antifungal agents were investigated by the micro-broth-dilution method in a study of Candida albicans. In the case of LF, its pepsin hydrolysate (LFhyd) or the LF-derived antimicrobial peptide Lactoferricin B (LF-B), the concentrations required to inhibit the growth of Candida decreased in the presence of relatively low concentrations of clotrimazole (CTZ). The minimum inhibitory concentration (MIC) of all azole antifungal agents tested was reduced by 1/4-1/16 in the presence of a sub-MIC level of each of these LF-related substances. Polyene and fluoropyrimidine antifungal agents did not show such a combined effect with these LF-related substances. The anti-Candida activity of LF or LF-B in combination with CTZ was shown to be synergistic by checkerboard analysis. These results indicate that LF-related substances function cooperatively with azole antifungal agents against C. albicans.

1. The immunomodulating activity of antifungal drugs was reviewed. Although results are conflicting, all azole drugs tend to be immunosuppressive, except for fluconazole, which has no immunologic effect. In contrast, the polyene antibiotic amphotericin B (AMPH) is immunostimulatory. 2. AMPH induced host resistance to Pseudomonas aeruginosa infection in mice, whereas no azole drugs did so. 3. Polymorphonuclear leukocytes are activated by AMPH, but not by any azole drugs, in terms of the level of their adherence. 4. No azole drugs induce in vitro tumor necrosis factor alpha (TNF) production by macrophages, whereas AMPH slightly but substantially does so. 5. AMPH potently primes macrophages in vitro and in vivo so that they produce large amounts of TNF after the secondary stimulation (triggering) by bacterial lipopolysaccharides or a streptococcal preparation used for antitumor immunotherapy, OK432. 6. Viable or heat-killed Candida albicans cells are capable of inducing in vitro TNF production by macrophages. This activity of the fungal cells is enhanced by AMPH.

Hydrosols of fresh leaves and rhizomes and air-dried rhizome of Zingiber officinale Roscoe contained citral as a major constituent. Geraniol was rich in the hydrosols of the steam-heated dry rhizome, and young leaves and rhizomes. These ginger hydrosols exhibited potent inhibitory activity against filamentous formation of Candida albicans, as compared with the hydrosols of other Zingiberaceae plants, Alpinia zerumbet and Zingiber mioga, as well as Mentha spicata used as a reference. Furthermore, ginger hydrosols showed the growth-inhibitory activity against C. albicans and Aspergillus fumigatus. The antifungal activity and citrus like flavor of the ginger hydrosols were ascribed to the major constituent, citral, which showed potent activity by itself. Key words: Zingiber officinale, hydrosol, volatile composition, filamentation inhibitory activity, Candida albicans, Aspergillus fumigatus